Project Summary Endothelial barrier is regulated at the level of adherens junctions (AJs), cell-cell adhesion structures mediated by the transmembrane protein VE-cadherin. Current model suggests that the tyrosine kinase c-Src functions as a negative regulator of endothelial barrier stimulating disassembly of AJs through phosphorylation of VE cadherin. However, our studies challenge this paradigm. We found that direct activation of Src using our engineered probe transiently enhances barrier function and induces formation of morphologically distinct AJs that exhibit reduced permeability. Our preliminary evidence suggests that Src-induced enhancement of endothelial barrier is mediated through signaling pathway regulating small GTPase Arf6. We also found that phosphorylation of VE cadherin on Tyr658/Tyr731 is critical for barrier strengthening by Src. Our studies also show that Src promotes formation of new contacts with extracellular matrix (focal adhesions) and stimulates interaction of focal adhesion protein p130Cas with VE cadherin. Based on these novel findings we hypothesize that Src signaling though Arf6 and VE-cadherin, and stimulation of new focal adhesions promote formation of AJs leading to strengthening of endothelial barrier. To define the role of each pathway downstream of Src, we propose to address the following questions. 1) We will determine the role of Arf6 activity in Src-stimulated formation of AJs, and identify Src-mediated pathways that activate Arf6. 2) We will define the role of Src signaling in AJs and the role of individual phosphorylation sites on VE-cadherin in formation of new AJs and enhancement of endothelial permeability. 3) We will determine the role of focal adhesions in Src-mediated enhancement of endothelial barrier and define the role of Src signaling through specific focal adhesion proteins. The timing and location of Src-mediated signaling is critical for regulation of AJs. Thus, to achieve precise temporal and spatial control of Src-mediated processes regulating AJs, we propose to employ engineered protein tools that will allow us to regulate precisely the activity of Src and phosphorylation of VE- cadherin in living cells. The level of control is unprecedented in that Src can be selectively activated and inactivated with tight temporal control and in specific subcellular locations in living cells. Importantly, Src activation can be restricted to a specific downstream targets and subcellular locations. Using these tools, we will dissect individual Src-mediated signaling pathways controlling assembly of AJs.